Rectoanal incontinence (RI) is one of the most debilitating gastrointestinal motility disorders affecting the elderly. A decrease in tone or stiffess of the smooth muscle of the internal anal sphincter (IAS) occurs with age and contributes to RI. Studies to date from our lab have provided insight into the regulatory control of IAS. In this competitive renewal application we propose to delineate novel control mechanisms of IAS muscle tone that can be targeted therapeutically. We will test the central hypothesis that miRNA regulation of RhoA/ROCK and SM22 is mechanistically linked to the age-dependent decrease in IAS tone and to RI, and that therapeutic targeting of miRNAs can increase IAS tone in vivo. We will establish differences in the signaling and contractile properties among multiple smooth muscle types of the lower GI tract; and in IAS as a function of age. Using both human and rat IAS, we propose studies to (Aim: 1) identify those intracellular signaling molecules that regulate IAS tone, and (Aim 2) identify miRNAs that regulate these intracellular signaling molecules, and develop strategies to reverse the miRNA-regulated decrease in IAS tone with age. We will establish multiple layers of control, focusing on the role of miRNAs as critical regulators of RhoA, RhoA-associated kinase (ROCK) and SM22. Preliminary biochemical and functional data support our hypothesis asserting the roles of RhoA, ROCK, and SM22 in determining SM tone among different SM types and age groups, while miRNA microarray analyses identify miR-139-5p and miR-1 as important regulators of ROCK and SM22 expression, respectively. Our robust approach includes cell, tissue, and in vivo models, enabling coordinated analysis of the miRNA-dependent proteome and signaling changes with functional effects. Beyond the unquestionable significance of the theme, the strengths of this proposal include: 1) exploration of novel mechanisms that explain the decrease in the IAS tone with age; 2) a comprehensive and coordinated omic, signaling and functional analysis that spans the reductionist- integrative spectrum; and 3) the development of a feasible therapeutic strategy for a disease that is currently untreatable.